A half-century or more has passed since the creation of a technique of culturing animal cells and the like in a test tube. Such a technique has been significantly developed together with the progress of science and technology.
In general, when a living animal is directly used for experiments, the results can be easily understood. However, such direct examination of a living animal has been problematic both technically and economically. Thus, a portion has been excised from an animal, and the cells thereof have been proliferated in an artificial environment such as in a Petri dish or in a test tube. This method is called a tissue culture method or a cell culture method. Since such a technique has not been difficult, it has become possible to produce pharmaceuticals, vaccines, diagnostic antigens, etc., by this method. For culturing animal cells in vitro, however, it is required to culture the cells almost under the same conditions as the original in vivo conditions. For example, conditions, such as an aseptic state or a temperature environment that is set at the same temperature as that in a living body, are applied.
Further, even if the aforementioned conditions have been satisfied, it has been necessary for cell division and proliferation to additionally supply a “cell growth factor” as a nutrient. Examples of such a cell growth factor include various types of hormones, insulin, putrescine, and a fibroblast growth factor. However, such cell growth factors have not yet been clarified in all cell species.
Accordingly, animal serums whose effect can be nonspecifically expected and which contain many “unknown” components are used instead of cell growth factors. Among such animal serums, bovine serum is selected because of a large bovine population and also because it can be stably supplied. Fetal calf serum has been frequently used because it contains only a small amount of toxic protein. In scientific studies, there are cases where a bovine-derived protein may be contained in a test material, although bovine is not an animal species of interest. However, the use of such a bovine-derived protein as a pharmaceutical for a human or other animal species may cause a problem.
The first problem is related to allergy. When a vaccine or a drug that contains bovine serum is parenterally injected into humans or non-bovine animals, a first injection may not cause a problem in many cases. However, a second injection or later injections may cause a problem regarding an allergy reaction. This phenomenon can be immunologically explained. That is, an animal only slightly reacts with a high-molecular substance (e.g. a protein having a molecular weight of 10,000 daltons or more) when the substance is exposed to the animal for the first time, and thus the administered substance is decomposed in vivo. However, a memory regarding exposure remains in immune system. Accordingly, when the same substance (antigen) is exposed to the animal for the second time or later, immunocytes that memorize the first exposure directly react with the substance, and as a result, a vital reaction that is stronger than that of the first exposure occurs in a short time. Depending on the types of humans or animals, there may be cases where they may have an unfavorable reaction with an antigen that is exposed from the outside. Such a reaction is typically referred to as an allergy reaction. Such an allergy reaction causes fever or swelling at an injected site, and in the worst case, humans or animals die from dyspnea due to respiratory obstruction and collapse.
The second problem is related to the contamination of pathogens or bovine serum antibodies contained in bovine serum. A famous example is contamination with Pestivirus, Retrovirus, Mycoplasma, etc. of bovine origin. Recently, prion that is a pathogen of bovine spongiform encephalopathy (BSE) known as mad cow disease has become problematic.
As stated above, although the use of bovine serum may cause troublesome occurrences, such bovine serum has commonly been used all over the world for production of vaccines particularly for use in a veterinary field that targets animals.
However, an attempt not to use bovine serum in the cell culture method (a serum-free medium (SFM) and a serum-free cell culture method) and the production of an experimental vaccine for bovines using such a serum-free medium and such a serum-free cell culture method have currently been reported (Makoschey et al., Serum-free produced bovine herpesvirus type 1 and bovine parainfluenza type 3 virus vaccines are efficacious and safe. Cytotechnology, 39: 139-145, 2002). In place of growth of cells in a cell culture medium from which serum components were removed, growth of cells in an existing medium, or a newly devised medium, to which various hormones and cell growth factors were added is disclosed in the following literatures:
Froud, S. J. The development, benefits and disadvantages of serum-free media. Brown, F., Cartwright, T., Horaud, F., Spieser, J. M. (eds): Animal sera, animal sera derivatives and substitutes used in the manufacture of pharmaceuticals; Viral safety and regulatory aspects. Dev. Biol. Stand., Basel, Karger, 1999, vol. 99, pp 157-166;
Merten, O.-W. Safety issues of animal products used in serum-freemedia. Brown, F., Cartwright, T., Horaud, F., Spieser, J. M. (eds): Animal sera, anima sera derivatives and substitutes used in the manufacture of pharmaceuticals: Viral safety and regulatory aspects. Dev. Biol. Stand., Basel, Karger, 1999, vol. 99, pp 167-180; and
Merten, O.-W. Development of serum-free media for cell growth and production of viruses/viral vaccines—Safety issues of animal products used in serum-free media. Brown, F., Hendriksen, C., Sesardic, D., Cussler, K. (eds): Advancing science and elimination of the use of laboratory animals for development and control of vaccines and hormones. Dev. Biol. Stand., Basel, Karger, 2002, vol. 111, pp 233-257.
Further, use of plant-derived components as medium additives is disclosed in the following literatures.
Noe et al., Fed-batch strategies for mammalian cell cultures. In, Spier, R. E., Griffiths, J. B., Berthold, W. (eds) Animal Cell Technology: products of Today, prospects for tomorrow, Oxford, Butterworth-Heinemann, 1994, pp 413-418.
Kazushi Shibuya et al.: Serum-free medium for animal cell culture, Published Japanese translation of PCT international publication for patent application (A), Japanese Patent Application Publication No. P2002-520014A (publication date: Jul. 9, 2002). This literature discloses that Chinese hamster ovary cells are cultured in a medium prepared by adding soybean protein hydrolysates, a yeast extract and optionally wheat protein hydrolysates to a uniquely prepared medium. As the soybean protein hydrolysates, various hydrolysates such as soluble polypeptides obtained by partial hydrolysis with a digestive enzyme are disclosed.
Kwon, S. M. et al. Use of plant-derived protein hydrolysates for enhancing growth of Bombyx mori (silkworm) insect cells in suspension culture. Biotechnol. Appl. Biochem., 42: 1-7, 2005. This literature discloses that plant-derived protein hydrolysates (trade name: HyPep 1510; Difco Co., Detroit, Mich., USA) can be used as a substitute for conventional bovine serum inculturing silkworm-derived insect cells, but does not disclose the composition of a medium used therein.
Chun, B.-H. et al. Use of plant hydrolysates for varicella virus production in serum-free medium. Biotechnol. Letters 27: 243-248, 2005. This literature discloses that human fetal lung cells (MRC-5) are cultured in a medium prepared by adding soybean protein hydrolysates etc., but not bovine serum, to a 2:1 mixture of Dulbecco's modified Eagle medium (DMEM) and Nutrient Mixture (Ham's F-12), and also that in this cell culture system, chickenpox virus is cultured. However, the medium used therein is compounded with recombinant human insulin, a recombinant human epidermal growth factor and a recombinant human fibroblast growth factor and contains animal proteins.
An animal protein-free medium for culturing cells (Baxter International Incorporated, Japanese Patent Application Publication No. P2005-532057A). This literature discloses a method of culturing cells by using a non-animal peptides derived from soybean hydrolysates and yeast hydrolysates, wherein the cells are cat-derived CRFK cells. However, the composition of a medium used therein is not disclosed.
As described above, a cat-derived cell strain produced without using animal-derived proteins, a medium used for culturing the same, and a method of multiplying a virus for use in feline vaccines, have not been disclosed. Further, a virus that infects cats will hardly multiply in other animal species without exception, and thus even if cells derived from other animals can be cultured without animal-derived proteins, such a technique cannot be applied to cat-derived cells.
Accordingly, there has been demand for cat-derived cells cultured in a medium not containing animal-derived proteins, an animal-derived protein-free medium for culturing the cells, safe vaccines and examination reagents for cats.